This invention is directed to aminooxy-modified nucleosides and oligonucleotides, to oligonucleotides that elicit RNase H for cleavage in a complementary nucleic acid strand, and to oligonucleotides wherein at least some of the nucleotides are functionalized to be nuclease resistant, at least some of the nucleotides of the oligonucleotide including a substituent that potentiates hybridization of the oligonucleotide to a complementary strand of nucleic acid, and at least some of the nucleotides of the oligonucleotide include 2xe2x80x2-deoxy-erythro-pentofuranosyl sugar moiety. The inclusion of one or more aminooxy moieties in such oligonucleotide provides, inter alia, for improved binding of the oligonucleotides to a complementary strand. The oligonucleotides and macromolecules are useful for therapeutics, diagnostics and as research reagents.
Oligonucleotides are known to hybridize to single-stranded RNA or single-stranded DNA. Hybridization is the sequence specific base pair hydrogen bonding of bases of the oligonucleotides to bases of target RNA or DNA. Such base pairs are said to be complementary to one another.
In determining the extent of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, denotes the temperature in degrees centigrade, at which 50% helical (hybridized) versus coil (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the bonds between the strands.
Oligonucleotides can be used to effect enzymatic cleavage of a target RNA by using the intracellular enzyme, RNase H. The mechanism of such RNase H cleavage requires that a 2xe2x80x2-deoxyribofuranosyl oligonucleotide hybridize to a target RNA. The resulting DNA-RNA duplex activates the RNase H enzyme and the activated enzyme cleaves the RNA strand. Cleavage of the RNA strand destroys the normal function of the RNA. Phosphorothioate oligonucleotides operate via this type of mechanism. However, for a DNA oligonucleotide to be useful for cellular activation of RNase H, the oligonucleotide must be reasonably stable to nucleases in order to survive in a cell for a time period sufficient for RNase H activation. For non-cellular uses, such as use of oligonucleotides as research reagents, such nuclease stability may not be necessary.
Several publications of Walder et al. describe the interaction of RNase H and oligonucleotides. Of particular interest are: (1) Dagle et al., Nucleic Acids Research 1990, 18, 4751; (2) Dagle et al., Antisense Research And Development 1991, 1, 11; (3) Eder et al., J. Biol. Chem. 1991, 266, 6472; and (4) Dagle et al., Nucleic Acids Research 1991, 19, 1805. According to these publications, DNA oligonucleotides having both unmodified phosphodiester internucleoside linkages and modified phosphorothioate internucleoside linkages are substrates for cellular RNase H. Since they are substrates, they activate the cleavage of target RNA by RNase H. However, the authors further note that in Xenopus embryos, both phosphodiester linkages and phosphorothioate linkages are also subject to exonuclease degradation. Such nuclease degradation is detrimental since it rapidly depletes the oligonucleotide available for RNase H activation.
As described in references (1), (2) and (4), to stabilize oligonucleotides against nuclease degradation while still providing for RNase H activation, 2xe2x80x2-deoxy oligonucleotides having a short section of phosphodiester linked nucleotides positioned between sections of phosphoramidate, alkyl phosphonate or phosphotriester linkages were constructed. Although the phosphoramidate-containing oligonucleotides were stabilized against exonucleases, in reference (4) the authors noted that each phosphoramidate linkage resulted in a loss of 1.6xc2x0 C. in the measured Tm value of the phosphoramidate containing oligonucleotides. Such a decrease in the Tm value is indicative of an decrease in hybridization between the oligonucleotide and its target strand.
Other authors have commented on the effect such a loss of hybridization between an oligonucleotide and its target strand can have. Saison-Behmoaras et al., EMBO Journal 1991, 10, 1111, observed that even though an oligonucleotide could be a substrate for RNase H, cleavage efficiency by RNase H was low because of weak hybridization to the mRNA. The authors also noted that the inclusion of
an acridine substitution at the 3xe2x80x2 end of the oligonucleotide protected the oligonucleotide from exonucleases.
U.S. Pat. No. 5,013,830, issued May 7, 1991, discloses mixed oligomers comprising an RNA oligomer, or a derivative thereof, conjugated to a DNA oligomer via a phosphodiester linkage. The RNA oligomers also bear 2xe2x80x2-O-alkyl substituents. However, being phosphodiesters, the oligomers are susceptible to nuclease cleavage.
European Patent application 339,842, filed Apr. 13, 1989, discloses 2xe2x80x2-O-substituted phosphorothioate oligonucleotides, including 2xe2x80x2-O-methylribooligonucleotide phosphorothioate derivatives. The above-mentioned application also discloses 2xe2x80x2-O-methyl phosphodiester oligonucleotides which lack nuclease resistance.
U.S. Pat. No. 5,149,797, issued Sep. 22, 1992, discloses mixed phosphate backbone oligonucleotides which include an internal portion of deoxynucleotides linked by phosphodiester linkages, and flanked on each side by a portion of modified DNA or RNA sequences. The flanking sequences include methyl phosphonate, phosphoromorpholidate, phosphoropiperazidate or phosphoramidate linkages.
U.S. Pat. No. 5,256,775, issued Oct. 26, 1993, describe mixed oligonucleotides that incorporate phosphoramidate linkages and phosphorothioate or phosphorodithioate linkages.
Although it has been recognized that cleavage of a target RNA strand using an oligonucleotide and RNase H would be useful, nuclease resistance of the oligonucleotide and fidelity of hybridization are of great importance in the development of oligonucleotide therapeutics. Accordingly, there remains a long-felt need for methods and materials that could activate RNase H while concurrently maintaining or improving hybridization properties and providing nuclease resistance. Such oligonucleotides are also desired as research reagents and diagnostic agents.
In accordance with one embodiment of this invention there are provided compounds of the structure: 
wherein:
T4 is Bx or Bxxe2x80x94L where Bx is a heterocyclic base moiety;
one of T1, T2 and T3 is L, hydrogen, hydroxyl, a protected hydroxyl or a sugar substituent group;
another one of T1, T2 and T3 is L, hydroxyl, a protected hydroxyl, a connection to a solid support or an activated phosphorus group;
the remaining one of T1, T2 and T3 is L, hydrogen, hydroxyl or a sugar substituent group provided that at least one of T1, T2, T3 and T4 is L or Bxxe2x80x94L;
said group L having one of the formulas; 
xe2x80x83wherein:
each m and mm is, independently, from 1 to 10;
y is from 1 to 10;
E is N(R1)(R2) or Nxe2x95x90C(R1)(R2);
each R1 and R2 is, independently, H, a nitrogen protecting group, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, wherein said substitution is OR3, SR3, NH3+, N(R3)(R4), guanidino or acyl where said acyl is an acid, amide or an ester;
or R1 and R2, together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O; and
each R3 and R4 is, independently, H, C1-C10 alkyl, a nitrogen protecting group, or R3 and R4, together, are a nitrogen protecting group;
or R3 and R4 are joined in a ring structure that optionally includes an additional heteroatom selected from N and O.
In some preferred embodiments, one of T1, T2 or T3 is L. In further preferred embodiments T3 is L.
In further preferred embodiments L is xe2x80x94Oxe2x80x94(CH2)2xe2x80x94Oxe2x80x94N(R1)(R2). In another preferred embodiment R1 is H or C1-C10 alkyl or C1-C10 substituted alkyl and R2 is Cl1-C10 substituted alkyl, preferably wherein R1 is C1-C10 alkyl and/or R2 is NH3+ or N(R3)(R4) C1-C10 substituted alkyl. In another preferred embodiment R1 and R2 are both C1-C10 substituted alkyl, with preferred substituents being independently, NH3+ or N(R3)(R4).
In some preferred embodiments Bx is adenine, guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine.
In some preferred embodiments R1 and R2 are joined in a ring structure that can include at least one heteroatom selected from N and O, with preferred ring structures being imidazole, piperidine, morpholine or a substituted piperazine wherein the substituent is prefereably C1-C12 alkyl.
In some preferred embodiments T1 is a protected hydroxyl. In other preferred embodiments T2 is an activated phosphorus group or a connection to a solid support. In some preferred embodiments, the solid support is microparticles. In further preferred embodiments the solid support material is CPG.
In some preferred embodiments L is bound to an exocyclic amino functionality of Bx. In other preferred embodiments, L is bound to a cyclic carbon atom of Bx.
In further preferred embodiments T4 is Bxxe2x80x94L. In still further preferred embodiments, Bx is adenine, 2-aminoadenine or guanine. In further preferred embodiments Bx is a pyrimidine heterocyclic base and L is covalently bound to C5 of Bx. In still further preferred embodiments Bx is a pyrimidine heterocyclic base and L is covalently bound to C4 of Bx. In yet further preferred embodiments Bx is a purine heterocyclic base and L is covalently bound to N2 of Bx. In still further preferred embodiments Bx is a purine heterocyclic base and L is covalently bound to N6 of Bx.
In accordance with some preferred embodiments, there are provided oligomeric compounds which incorporate at least one nucleosidic compound that is functionalized to increase nuclease resistance of the oligomeric compounds. In a further embodiment oligomeric compounds are functionalized with a substituent group to increase their binding affinity to target RNAs.
The oligomeric compounds preferably comprise a plurality of nucleoside units of the structure: 
wherein:
T4 of each nucleoside unit is, independently, Bx or Bxxe2x80x94L where Bx is a heterocyclic base moiety;
one of T5, T6 and T7 of each nucleoside unit is, independently, L, hydroxyl, a protected hydroxyl, a sugar substituent group, an activated phosphorus group, a connection to a solid support, a nucleoside, a nucleotide, an oligonucleoside or an oligonucleotide;
another of T5, T6 and T7 of each nucleoside unit is, independently, a nucleoside, a nucleotide, an oligonucleoside or an oligonucleotide;
the remaining one of T5, T6 and T7 of each nucleoside unit is, independently, is L, hydrogen, hydroxyl, a protected hydroxyl, or a sugar substituent group;
provided that on at least one of said nucleoside units T4 is Bxxe2x80x94L or at least one of T5, T6 and T7 is L;
said group L having one of the formulas; 
xe2x80x83wherein:
each m and mm is, independently, from 1 to 10;
y is from 1 to 10;
E is N(R1)(R2) or Nxe2x95x90C(R1)(R2)
each R1 and R2 is, independently, H, a nitrogen protecting group, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, wherein said substitution is OR3, SR3, NH3+, N(R3)(R4), guanidino or acyl where said acyl is acid, amide or ester,
or R1 and R2, together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O; and
each R3 and R4 is, independently, H, C1-C10 alkyl, a nitrogen protecting group, or R3 and R4, together, are a nitrogen protecting group or wherein R3 and R4 are joined in a ring structure that optionally includes an additional heteroatom selected from N and O.
In some preferred embodiments of the oligomeric compounds of the invention, at least one of T1, T2 or T3 is L. In further preferred embodiment, at least one T3 is L.
In further preferred embodiments of the oligomeric compounds of the invention, at least one L is xe2x80x94Oxe2x80x94(CH2)2xe2x80x94Oxe2x80x94N(R1)(R2). In further preferred embodiments of the oligomeric compounds of the invention, R1 is H or C1-C10 alkyl or C1-C10 substituted alkyl and R2 is C1-C10 substituted alkyl, preferably wherein R1 is C1-C10 alkyl and/or R2 is NH3+ or N(R3)(R4) C1-C10 substituted alkyl. In still further preferred embodiments of the oligomeric compounds of the invention, R1 and R2 are both C1-C10 substituted alkyl, with preferred substituents being independently, NH3+ or N(R3)(R4)
In some preferred embodiments of the oligomeric compounds of the invention, Bx is adenine, guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine.
In some preferred embodiments of the oligomeric compounds of the invention, R1 and R2 are joined in a ring structure that can include at least one heteroatom selected from N and O, with preferred ring structures being imidazole, piperidine, morpholine or a substituted piperazine wherein the substituent is preferably C1-C12 alkyl.
In some preferred embodiments of the oligomeric compounds of the invention, T1 is a protected hydroxyl. In other preferred embodiments of the oligomeric compounds of the invention, T2 is an activated phosphorus group or a connection to a solid support. In some preferred embodiments of the oligomeric compounds of the invention, the solid support is microparticles. In further preferred embodiments the solid support material is CPG.
In some preferred embodiments of the oligomeric compounds of the invention, L is bound to an exocyclic amino functionality of Bx. In other preferred embodiments of the oligomeric compounds of the invention, L is bound to a cyclic carbon atom of Bx.
In further preferred embodiments of the oligomeric compounds of the invention, T4 is Bxxe2x80x94L. In still further preferred embodiments, Bx is adenine, 2-aminoadenine or guanine. In further preferred embodiments of the oligomeric compounds of the invention, Bx is a pyrimidine heterocyclic base and L is covalently bound to C5 of Bx. In still further preferred embodiments of the oligomeric compounds of the invention, Bx is a pyrimidine heterocyclic base and L is covalently bound to C4 of Bx. In yet further preferred embodiments of the oligomeric compounds of the invention, Bx is a purine heterocyclic base and L is covalently bound to N2 of Bx. In still further preferred embodiments of the oligomeric compounds of the invention, Bx is a purine heterocyclic base and L is covalently bound to N6 of Bx.
In some preferred embodiments of the oligomeric compounds of the invention, the oligomeric compounds are from 5 to 50 nucleoside units in length. In further preferred embodiments of the oligomeric compounds of the invention, the oligomeric compounds are from 8 to 30 nucleoside units in length, with 15 to 25 nucleoside units in length being more pporeferred.
In some preferred embodiments, chimeric oligomeric compounds are provided that are specifically hybridizable with DNA or RNA comprising a sequence of linked nucleoside units. Preferably, the sequence is divided into a first region having linked nucleoside units and a second region being composed of linked nucleoside units having 2xe2x80x2-deoxy sugar moieties. The linked nucleoside units of at least one of the first or second regions are connected by phosphorothioate linkages and at least one of the linked nucleoside units of the first region bears a group L that is covalently attached to the heterocyclic base or the 2xe2x80x2, 3xe2x80x2 or 5xe2x80x2 position of the sugar moiety wherein the group L has one of the formulas: 
where
each m and mm is, independently, from 1 to 10;
y is from 1 to 10;
E is N(R1)(R2) or Nxe2x95x90C(R1)(R2);
each R1 and R2 is, independently, H, a nitrogen protecting group, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, wherein the substitution is OR3, SR3, NH3+, N(R3)(R4), guanidino or acyl where the acyl is an acid, amide or an ester;
or R1 and R2, together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O; and
each R3 and R4 is, independently, H, C1-C10 alkyl, a nitrogen protecting group, or R3 and R4, together, are a nitrogen protecting group; and
or R3 and R4 are joined in a ring structure that optionally includes an additional heteroatom selected from N and O.
In some preferred embodiments, the nucleoside units of the first and second regions are connected by phosphorothioate internucleoside linkages. In further preferred embodiments, the nucleoside units of the first region are connected by phosphodiester internucleoside linkages and the nucleoside units of the second region are connected by phosphorothioate internucleoside linkages. In still further preferred embodiments, the nucleoside units of the first region are connected by phosphorothioate internucleoside linkages and the nucleoside units of the second region are connected by phosphodiester internucleoside linkages.
In some preferred embodiments, the second region has at least three nucleoside units. In further preferred embodiments, the second region has at least five nucleoside units.
In some preferred embodiments, the chimeric oligomeric compound has a third region having 2xe2x80x2-O-alkyl substituted nucleoside units, wherein the second region is positioned between the first and third regions. In further preferred embodiments, the nucleoside units of the first, second and third regions are connected by phosphorothioate linkages. In further preferred embodiments, the nucleoside units of the first and third regions are connected by phosphodiester linkages and the nucleoside units of the second region are connected by phosphorothioate linkages. In another preferred embodiment, the nucleoside units of the first and third regions are connected by phosphorothioate linkages and the nucleoside units of the second region are connected by phosphodiester linkages.
In some preferred embodiments, the second region has at least three nucleoside units. In further preferred embodiments, the second region has at least five nucleoside units.
In some preferred embodiments, at least one of the 2xe2x80x2-O-alkyl substituted nucleoside units of the third region bears an L group.
The nucleotides forming oligonucleotides of the present invention can be connected via phosphorus linkages. Preferred phosphorous linkages include phosphodiester, phosphorothioate and phosphorodithioate linkages, with phosphodiester and phosphorothioate linkages being particularly preferred.